Article

Recommended Mass Spectrometry-Based Strategiesto Identify Ricin-Containing Samples

Suzanne R. Kalb 1,†, David M. Schieltz 1,†, François Becher 2,†, Crister Astot 3,†,Sten-Åke Fredriksson 3,† and John R. Barr 1,*

Received: 25 June 2015; Accepted: 24 August 2015; Published: 25 November 2015Academic Editor: Andreas Rummel and Brigitte G. Dorner

1 Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA;skalb@cdc.gov (S.R.K.); dschieltz@cdc.gov (D.M.S.)

2 Service de Pharmacologie et d’Immunoanalyse, Institut de Biologie et de Technologies de Saclay (iBiTec-S),Commissariat à l’Énergie Atomique et aux Énergies Alternatives (CEA), 91191 Gif-sur-Yvette, France;francois.becher@cea.fr

3 The Swedish Defence Research Agency (FOI), SE-901 82 Umeå, Sweden; crister.astot@foi.se (C.A.);sten-ake.fredriksson@foi.se (S.-A.F.)

* Correspondence: jbarr@cdc.gov; Tel.: +1-770-488-7848; Fax: +1-770-488-0509† These authors contributed equally to this work.

Abstract: Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) togetherwith a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assayshave the capacity to unambiguously identify ricin and to detect ricin’s activity in samples withcomplex matrices. These qualitative and quantitative assays enable detection and differentiationof ricin from the less toxic RCA120 through determination of the amino acid sequence of theprotein in question, and active ricin can be monitored by MS as the release of adenine from thedepurination of a nucleic acid substrate. In this work, we describe the application of MS-basedmethods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied aspart of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samplescontaining ricin or RCA120 with the exception of the sample spiked with the lowest concentration(0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricinand RCA120 based on amino acid determination. Mass spectrometric methods were also successfulat ranking the functional activities of the samples, successfully yielding semi-quantitative results.These results indicate that MS-based assays are excellent techniques to detect, differentiate, andquantify ricin and RCA120 in complex matrices.

Keywords: ricin; RCA120; Ricinus communis; mass spectrometry

1. Introduction

Ricin is a protein toxin produced by the castor bean plant, Ricinus communis. It is composedof two polypeptide chains, known as the A- and B-chains, which are each approximately 32 kDa,making intact ricin approximately 64 kDa with glycosylation [1]. Ricin belongs to the type 2ribosome-inactivating protein toxins (RIP-II toxins). The A-chain of ricin has a specific enzymaticactivity, as it depurinates a single adenosine that is part of a GAGA tetraloop of 28S ribosomalRNA [2,3]. This prevents the binding of elongation factor 2 (EF-2) to the ribosome, leading to thecessation of protein synthesis [4]. The termination of protein synthesis causes the severe physicaleffects of the ricin toxin and can lead to death. The B-chain has lectin activity and brings theenzymatically active A-chain to its target through cell receptor binding and endocytosis [5]. Both

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Toxins 2015, 7, 4881–4894

the A and B-chains contain N-linked glycosylation [6,7], and although both chains are needed forin vivo toxicity, glycosylation of only the B-chain is needed for maximum toxicity [8].

R. communis also produces a closely related protein known as RCA120, or agglutinin. RCA120 isa heterodimer with two A-chains and two B-chains linked by one disulfide bond between Cys156 ofeach A-chain and has an intact mass of approximately 120 kDa. It has the same enzymatic activity asricin, but is orders of magnitude less active than ricin [9]. These two proteins are approximately 89%identical in amino acid sequence, as seen in Figure 1, which makes the two proteins quite similar;in fact, many antibodies have cross-reactivity between ricin and RCA120 [10]. Nonetheless, the 11%difference can be used for differentiation between the two proteins, provided that an appropriateanalytical technique is utilized.

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described/compiled in the following sections—some of these methods have been applied to the analysis

of nine blinded samples supplied as part of the EQuATox international ricin proficiency test [14]. The

aim was to test the participating laboratories’ capacity to identify the presence or absence of ricin or

RCA120 in the blinded samples, consisting of stabilizing buffer, milk, meat extract and fertilizer as seen

in Table 1. Furthermore, for the samples, which were found to contain a toxin, differentiation between

ricin and RCA120 was desired. The final goals were to quantify the amount of toxin in the samples and

to provide a ranking of functional activity of three specified samples.

Figure 1. Amino acid sequence alignment of ricin D, ricin E and RCA120. Amino acids

which differ are noted in the consensus line as an empty space or as “+” if the changes are

conserved. The A-chain is depicted in blue and the B-chain in green. Peptides used for

targeted mass spectrometric identification/differentiation are underlined. Highlighted

cysteines indicate disulfide bond formation.

Ricin D MYAVATWLCFGSTSGWSFTLEDNNIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRRicin E MYAVATWLCFGSTSGWSFTLEDNNIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRRCA120 MYAVATWLCFGSTSGWSFTLEDNNIFPKQYPIINFTTADATVESYTNFIRAVRSHLTTGADVRHEIPVLPNRVGLPISQRConsensus MYAVATWLCFGSTSGWSFTLEDNNIFPKQYPIINFTTA ATV+SYTNFIRAVR LTTGADVRHEIPVLPNRVGLPI+QR  Ricin D FILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQLAGNLRENRicin E FILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQLAGNLRENRCA120 FILVELSNHAELSVTLALDVTNAYVVGCRAGNSAYFFHPDNQEDAEAITHLFTDVQNSFTFAFGGNYDRLEQLGG-LRENConsensus FILVELSNHAELSVTLALDVTNAYVVG RAGNSAYFFHPDNQEDAEAITHLFTDVQN +TFAFGGNYDRLEQL G LREN  Ricin D IELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTRicin E IELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTRCA120 IELGTGPLEDAISALYYYSTCGTQIPTLARSFMVCIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTConsensus IELG GPLE+AISALYYYST GTQ+PTLARSF++CIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLST

 Ricin D AIQESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALMVYRCAPPPSSQFSLLIRPVVPNFNADVCMDPEPIVRIVGRNRicin E AIQESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALMVYRCAPPPSSQFSLLIRPVVPNFNADVCMDPEPIVRIVGRNRCA120 AIQESNQGAFASPIQLQRRNGSKFNVYDVSILIPIIALMVYRCAPPPSSQFSLLIRPVVPNFNADVCMDPEPIVRIVGRNConsensus AIQESNQGAFASPIQLQRRNGSKF+VYDVSILIPIIALMVYRCAPPPSSQFSLLIRPVVPNFNADVCMDPEPIVRIVGRN  Ricin D GLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVYVMIYDCNTAATDATRWQIWDNGTRicin E GLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYPSGVYVMIYDCNTAATDATRWQIWDNGTRCA120 GLCVDVTGEEFFDGNPIQLWPCKSNTDWNQLWTLRKDSTIRSNGKCLTISKSSPRQQVVIYNCSTATVGATRWQIWDNRTConsensus GLCVDV F +GN IQLWPCKSNTD NQLWTL++D+TIRSNGKCLT V+IY+C+TA ATRWQIWDN T  Ricin D IINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYARicin E IINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGMCLQANSGKVWLEDCSSEKAEQQWALYARCA120 IINPRSGLVLAATSGNSGTKLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGMCLQANSGKVWLEDCTSEKAEQQWALYAConsensus IINPRS LVLAATSGNSGT LTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYG+CLQANSG+VW+EDC+SEKAEQQWALYA

 Ricin D DGSIRPQQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRASDPSLKQIILYPLHGDPNQIRicin E DGSIRPQQNRDNCLTTDANIKGTVVKILSCGPASSGQRWMFKNDGTILNLYNGLVLDVRRSDPSLKQIIVHPVHGNLNQIRCA120 DGSIRPQQNRDNCLTTDANIKGTVVKILSCGPASSGQRWMFKNDGTILNLYNGLVLDVRRSDPSLKQIIVHPFHGNLNQIConsensus DGSIRPQQNRDNCLT+D+NI+ TVVKILSCGPASSGQRWMFKNDGTILNLY+GLVLDVR SDPSLKQII++P HG+ NQI

 Ricin D WLPLF 565 Ricin E WLPLF RCA120 WLPLF Consensus WLPLF 

Figure 1. Amino acid sequence alignment of ricin D, ricin E and RCA120. Amino acids whichdiffer are noted in the consensus line as an empty space or as “+” if the changes are conserved.The A-chain is depicted in blue and the B-chain in green. Peptides used for targeted massspectrometric identification/differentiation are underlined. Highlighted cysteines indicate disulfidebond formation.

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Table 1. Identity of EQuATox samples with qualitative results using mass spectrometric (MS/MS) analysis. Signature trypsin digest peptides of ricin D(Uniprot P02879).

Sample # S1 S2 S3 S4 S5 S6 S7 S8 S9

Matrix 0.1% BSAin PBS

0.1% BSAin PBS

0.1% BSAin PBS

Skimmedmilk

0.1% BSAin PBS

0.1% BSAin PBS

0.1% BSAin PBS Meat extract Organic fertilizer

Nominal toxin N/A RCA120 Ricin Ricin RCA120 Ricin Ricin Ricin Ricin, RCA120Conc. N/A 572851 504 473 445 589508 0.414 484 306, 42Unit µg/L µg/L µg/L µg/L µg/L µg/L µg/L mg/kg

Observed toxin RCA120 Ricin Ricin,RCA120 RCA120 Ricin Ricin Ricin Ricin, RCA120

Observed toxin activity ´ + + + + + ´ + +Ricin D (Uniprot P02879) signature trypsin digest peptides

T# Amino Acid Sequence S1 S2 S3 S4 S5 S6 S7 S8 S9T2a QYPIINFTTAGATVQSYTNFR ˝

T5a LTTGADVR � � � � � �

T7a VGLPINQR � � � � � � � � ˝ �

T9a AGNSAYFFHPDNQEDAEAITHLFTDVQNRT10a YTFAFGGNYDR ˝

T11a LEQLAGNLR ˝

T23a FSVYDVSILIPIIALMVYR

T3b-T5b NGLCVDVR(-ss-)FHNGNAIQLWPCK ˝ ˝ ˝

T6b SNTDANQLWTLK ˝

T11b WQIWDNGTIINPR ˝

T14b-T16b DNCLTSDSNIR (-ss-)ILSCGPASSGQR ˝ ˝ ˝ ˝

T18b NDGTILNLYSGLVLDVR ˝ ˝ ˝ ˝ ˝

T19b ASDPSLK � � � � �

T20b QIILYPLHGDPNQIWLPLF

�: Identified by targeted liquid chromatography LC-MS/MS; ˝: Identified by non-targeted LC-MS/MS and : Confirmed by LC-MS/MS.

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In addition, there are two ricin isoforms, D and E, which differ in their B-chain sequence. RicinE is thought to have evolved through a gene recombination between the ricin D and the RCA120genes [11]. Thus, the B-chain of ricin E shares its N-terminus with ricin D and the C-terminus withRCA120 as seen in Figure 1. Ricin E is produced in some of the R. communis ecotypes studied and hasonly been detected together with ricin D. Specific differentiation of the ricin isoforms from RCA120is critical for forensic or epidemiological purposes, and the close similarity between the proteinshas made it hard to produce specific antibodies (Ab) without a high cross-reactivity between theproteins [10]. At least one specific immunoassay based on monoclonal Ab has been reported [12] butthe availability has generally been a limiting factor for the use of immunological methods for ricinspecific detection. However, the sequence differences in ricin and RCA120 can be monitored by massspectrometry to allow their identification.

Mass spectrometry (MS) is an instrumental measurement technique with widespread use inlife sciences. The MS technique has been described in detail elsewhere [13]. A wide variety ofinstrumental methods involving MS and MS/MS measurements have been successful in detecting,differentiating and quantifying ricin and RCA120 in complex matrices. The different possibleanalytical strategies will be described/compiled in the following sections—some of these methodshave been applied to the analysis of nine blinded samples supplied as part of the EQuAToxinternational ricin proficiency test [14]. The aim was to test the participating laboratories’ capacity toidentify the presence or absence of ricin or RCA120 in the blinded samples, consisting of stabilizingbuffer, milk, meat extract and fertilizer as seen in Table 1. Furthermore, for the samples, which werefound to contain a toxin, differentiation between ricin and RCA120 was desired. The final goals wereto quantify the amount of toxin in the samples and to provide a ranking of functional activity of threespecified samples.

2. Mass Spectrometric (MS) Techniques for Analysis of Ricin

2.1. Determination of Molecular Weight of the Intact Ricin Protein by Mass Spectrometric Analysis

The simplest technique for analysis of ricin by mass spectrometry is to detect the intact ricinprotein with an approximate molecular weight of 64 kDa. Thus, the detection of a signal with amass close to 64 kDa could indicate the presence of ricin. This technique was first reported in 2000using electrospray mass spectrometry for ricin characterization [15]. Electrospray ionization (ESI) ofintact ricin produced multiply charged ions, and when the data were de-convoluted, a pattern ofpeaks corresponding to several glycoforms with a median molecular weight of approximately 63 kDawas generated. MALDI-TOF mass spectrometry has also been reported for the analysis of intactricin and this work reported a molecular weight of approximately 62,766 Da [16]. In addition toanalysis on purified ricin, matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF/MS) of intact ricin was also reported using crude extracts from castor beans [17].

Although analysis of intact ricin can provide useful information including differentiationbetween ricin and RCA120, it is not a definitive analysis of ricin, as there are many proteins, whichhave the same approximate molecular weight as ricin. Additionally, this method suffers from thehigh limit of detection of concentrations greater than 1 µg/mL [17]. Nonetheless, in combinationwith other detection methods with higher specificity, this analysis can provide useful information.

2.2. MS Analysis of the Digested Ricin Protein

Another mass spectrometric technique for ricin identification is peptide mass fingerprinting(PMF) in which the ricin is digested into peptides before analysis by high resolution MS, yieldingaccurate mass data of the peptides. This allows for protein identification based on the match ofacquired peptide masses with the theoretical peptide masses derived in-silico. The first applicationof peptide mass fingerprinting to identify ricin was reported in 2001 [16] where ricin digestedwith trypsin was analyzed with both MALDI-TOF MS and ESI. Eleven peaks corresponding to the

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A- and B-chains or ricin were detected by ESI mass spectrometry, and fourteen were visible byMALDI-TOF MS. A similar study in 2006 generated comparable results, with 17 peptides originatingfrom ricin [18], and another study in 2008 reported observation of 20 peptides with a limit of detectionof 50 ng/mL [19]. The digestion process was significantly shortened first in 2008 [19] and then in2010 using on-target tryptic digestion of 30 min to generate 16 peptides from ricin, and some of thepeptides could be used to differentiate ricin from RCA120 [20].

Although PMF analyses produce useful information with the potential to differentiate betweenricin and RCA120, it is not a conclusive analysis of ricin, as the molecular weight of the peptidescarries no information as to their amino acid sequences. In conjunction with other detection methods,PMF can yield useful information in a short timeframe, and the information obtained from thistype of analysis can be more informative and is certainly more sensitive than measurements on theintact protein.

3. MS/MS Techniques for Analysis of Ricin

3.1. Targeted MS/MS Approach

In order to increase the selectivity and sensitivity of the peptide analysis described above,tandem mass spectrometry has been used in targeted approaches for the detection of specific peptides.The operation of triple quadrupole or ion trap mass spectrometers to monitor specific transitionsafter collision induced dissociation (CID) from parent peptide ions selected in the first MS stage, toproduct (fragment) ions selected in the second MS stage, allows an analysis of ricin peptides withmuch higher selectivity than the MS-methods described above. This type of approach is highlytargeted and generally achieves detection of a peptide at a lower limit of detection than other types ofmass spectrometers, as seen in a 2011 report in which this approach was used to analyze ricin spikedinto complex matrices [21]. By focusing on both the A-chain and the B-chain peptides with aminoacid sequences unique for ricin, a selective method with an extraordinarily low limit of detection of0.64 ng/mL was achieved. This approach was later reported for the study of ricin in 18 differentR. communis cultivars [22].

Targeted MS/MS of ricin was used by at least one laboratory as part of the EQuATox study, andthe ricin protein was found in samples S3, S4, S6, S8 and S9 as seen in Table 1. These data show thatthe targeted MS/MS analysis of ricin was correct, with the exception of the lowest level sample (S7)which was below the detection limit of the method, and that this analytical approach yielded excellentqualitative results for ricin. This process was also used to determine the presence of RCA120 in thenine blinded samples. From these analyses, it was determined that RCA120 was present in samplesS2, S4, S5 and S9, as seen in Table 2. It was later revealed that sample S4 contained only ricin and didnot contain RCA120. Because RCA120 is several orders of magnitude less toxic than ricin, there hasbeen more of an emphasis on detection of ricin rather than RCA120. Entities have chosen to developand validate assays for ricin detection, often including RCA120 detection as an afterthought.

Nonetheless, a targeted MS/MS approach remains one of the best methods to detect anddifferentiate ricin and RCA120 with the ability to distinguish even between ricin D and ricin E,provided that the correct peptide ions are selected for monitoring.

3.2. Proteomic MS/MS Approach

The use of scanning tandem mass spectrometers (e.g., quadrupole-time-of-flight (QTOF) MS)allows for the detection of a full spectrum of product ions from a peptide parent ion selected forMS/MS analysis, as described above. As peptide ions can be induced to systematically fragmentalong the peptide backbone inside the tandem mass spectrometer, the formation of the fragment ionsis a controlled process, allowing for determination of amino acid sequences by examining the massdifferences of the fragment ions [23]. The use of liquid chromatography (LC) retention time, mass ofintact peptide and masses of fragment ions can be used in concert to verify the presence of a particular

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peptide, which can then be used to verify the existence of a particular protein, provided that the aminoacid sequence of that peptide is unique.

MS/MS amino acid sequencing of ricin was first reported in 2005 [24]. In this work, ricin wasextracted from castor beans and digested with trypsin with the resultant tryptic peptides analyzedwith a QTOF mass spectrometer. This yielded fourteen peptides from the A-chain of ricin and sixteenpeptides from the B-chain, in addition to peptides specific for RCA120 [22]. Another similar methodincludes a shortened digestion time without prior reduction of disulfide bonds, in which the peptidesfrom the A-chain, peptides from the B-chain and the peptide containing the intact interchain disulfidebond verifying the presence of the link between the A- and B-chains were identified and differentiatedfrom RCA120 [25]. This approach was reported for the identification of ricin spiked into complexmatrices such as milk, apple juice, serum and saliva [26], in surface swabs from a public healthinvestigation [27], as well as identification of ricin and abrin in a forensic investigation [28].

Table 2. RCA120 trypsin digest peptides (chain A: Uniprot P06750, chain B: GI: 225114). Peptidesdistinguishing RCA120 from ricin are shown in bold type face. Peptides in italics are also found inricin E (GI: 225419).

Sample # S1 S2 S3 S4 S5 S6 S7 S8 S9

T2a QYPIINFTTADATVESYTNFIRT4a SHLTTGADVR � � � � � � �T5a HEIPVLPNR � � � � � � � ˝ �T6a VGLPISQR � � � � � � � �T9a LEQLGGLR ˝

T12a FQYIEGEMR ˝

T17a SAPDPSVITLENSWGR ˝

T18a LSTAIQESNQGAFASPIQLQR ˝

T22a-T1b CAPPPSSQF(ss-)ADVCMDPEPIVR ˝

T3b NGLCVDVFGEEFTDGNPIQLWPCK ˝ ˝ ˝

T4b SNTDWNQLWTLR

T8b-T9b CLTISK(-ss-)SSPGQQVVIYNCSTATVGATR ˝ ˝

T10b WQIWDNR ˝ ˝ ˝

T11b TIINPTSGLVLAATSGNSGTKT14b AEQQWALYADGSIRPQQNR ˝ ˝ ˝

T15b-T17b DNCLTTDANIK(-ss-)ILSCGPASSGQR ˝ ˝ ˝ ˝ ˝ ˝

T19b NDGTILNLYNGLVLDVR ˝ ˝ ˝ ˝ ˝ ˝

T22b QIIVHPVHGNLNQIWLPLF ˝ ˝

�: Identified by targeted LC-MS/MS; ˝: identified by non-targeted LC-MS and : confirmed by LC-MS/MS.

Tables 1 and 2 indicate the peptides detected by targeted LC-MS/MS on a triple quadrupoleinstrument and by non-targeted LC-high resolution accurate mass MS on a QTOF instrument. Thesequence coverage obtained by the non-targeted approach was approximately 70% in the highconcentration samples S2 and S6, while in the complex and lower concentration samples S4 and S8 itwas 30%–50%. The toxins were unambiguously identified by subsequent analysis by time-scheduledLC-MS/MS of selected peptides indicated in Tables 1 and 2. Figure 2A shows chromatograms of theLC-MS analysis of the digest of sample S4. Diagnostic ricin peptides are indicated by filled peaks. Theoffscale peaks in the base peak chromatogram (grey trace) are from trypsin digest peptides of milkproteins not removed in the sample preparation procedure. Figure 2B shows the close resemblancebetween the product ion spectrum of m/z 507.29 in sample S4 and T11a in the ricin reference digest.

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Figure 2. LC-high resolution accurate mass MS analysis of the trypsin digest of a 200 μL

aliquot of immunopurified sample S4. (A) Base peak chromatogram (grey) overlaid on

extracted ion chromatograms of diagnostic ricin D and E peptides (20 ppm mass window).

(B) Product ion spectrum of m/z 507.29 at retention time 14.7 min in the S4 digest (upper)

compared to the product ion spectrum of T11 of chain A in the ricin reference digest (lower).

The LC-MS/MS technique is one of the few techniques, which can effectively differentiate ricin from

RCA120. Although a wide variety of techniques such as enzyme-linked immunosorbant assay (ELISA),

cytotoxicity, Western blots, gel electrophoresis, nuclear magnetic resonance (NMR), lateral flow assay,

hemagglutination and mass spectrometry were used to analyze the samples in this EQuATox study, most

methods could not differentiate between ricin and RCA120. LC-MS/MS techniques were the most

effective at correctly differentiating high concentrations of ricin and RCA120 with success rates of

79%–87% when looking at sample S2 with a high concentration of RCA120 and sample S6 with a high

concentration of ricin as seen in Table 3 and described in a separate publication in this special issue [14].

Table 3. Success rates for various methods of differentiation of ricin and RCA120 of the

two highest concentration samples.

Sample Principle Mean Success Rate

S2 Mass spectrometry 79% S2 Functional method 57% S2 Immunological method 43% S6 Mass spectrometry 87% S6 Immunological method 71% S6 Functional method 43%

While the EQuATox program did not ask for differentiation beyond ricin and RCA120, LC-MS/MS

methods have proven effective at an extended level of differentiation. Differentiation between ricin and

Figure 2. LC-high resolution accurate mass MS analysis of the trypsin digest of a 200 µL aliquotof immunopurified sample S4. (A) Base peak chromatogram (grey) overlaid on extracted ionchromatograms of diagnostic ricin D and E peptides (20 ppm mass window). (B) Product ionspectrum of m/z 507.29 at retention time 14.7 min in the S4 digest (upper) compared to the production spectrum of T11 of chain A in the ricin reference digest (lower).

The LC-MS/MS technique is one of the few techniques, which can effectively differentiatericin from RCA120. Although a wide variety of techniques such as enzyme-linked immunosorbantassay (ELISA), cytotoxicity, Western blots, gel electrophoresis, nuclear magnetic resonance (NMR),lateral flow assay, hemagglutination and mass spectrometry were used to analyze the samples inthis EQuATox study, most methods could not differentiate between ricin and RCA120. LC-MS/MStechniques were the most effective at correctly differentiating high concentrations of ricin andRCA120 with success rates of 79%–87% when looking at sample S2 with a high concentration ofRCA120 and sample S6 with a high concentration of ricin as seen in Table 3 and described in a separatepublication in this special issue [14].

Table 3. Success rates for various methods of differentiation of ricin and RCA120 of the two highestconcentration samples.

Sample Principle Mean Success Rate

S2 Mass spectrometry 79%S2 Functional method 57%S2 Immunological method 43%S6 Mass spectrometry 87%S6 Immunological method 71%S6 Functional method 43%

While the EQuATox program did not ask for differentiation beyond ricin and RCA120,LC-MS/MS methods have proven effective at an extended level of differentiation. Differentiationbetween ricin and RCA120 or between ricin D and ricin E can be very important for forensic orepidemiological purposes, and such a distinction is often necessary for law enforcement reasons. Bothricin isoforms were present in the EQuATox spiking material and ricin D-specific peptides T14b-T16b

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disulfide connected peptide, T18b, T19b and T20b were detected (Table 1). However, differentiationof ricin E in the presence of the D isoform and RCA120 is complicated by the complete overlap ofthe amino acid sequence with either the D isoform or with RCA120. The presence of the E isoformcan be determined by quantification of the sum of ricin E and RCA120 using the E/RCA120 sharedpeptides, subtracted by the amount of RCA120 using the RCA120-specific peptides and calibrationsamples of a purified RCA120 standard. Alternatively, the D and E isoforms can be separated usingion-exchange chromatography [24], so it is possible that this further differentiation could have beenmade as part of the EQuATox study if it was in question.

4. MS and MS/MS Techniques for Analysis of Ricin’s Enzymatic Activity

4.1. Importance of Ricin Activity Measurements

As mentioned previously, ricin has a specific in vivo enzymatic activity. It depurinates anadenosine which is part of the GAGA tetraloop of 28S ribosomal RNA, thereby halting proteinsynthesis. Several laboratories have shown that the enzymatic activity of the A-chain of ricin canbe monitored by mass spectrometry. Typically, this process involves incubation of the protein ofinterest with a shortened mimic of 28S ribosomal RNA, which contains the GAGA tetraloop. Ricinwill depurinate the GAGA tetraloop, resulting in the release of free adenine and a decrease in massof the 28S ribosomal RNA mimic. This assessment of the functionality of the A-chain of ricin isimportant for anyone desiring to understand the health threat of ricin. However, there are a numberof ribosome inactivating proteins (including RCA120) with the same enzymatic activity of ricin, sosuch detection methods work best with supplementation or in combination with another method toaddress specificity and/or by the use of functionally blocking antibodies [29].

4.2. MS Analysis of Ricin Activity Measurements

Although ricin may be subject to denaturation, especially in food samples, the decrease in massof the 28S ribosomal RNA mimic can be monitored by mass spectrometry, allowing for an accuratedetermination of the enzymatic activity of the A-chain of ricin, as seen in Figure 3. One successfulexample of this method involves a 2009 report in which ricin’s enzymatic activity was detected usingMALDI-TOF MS to detect the depurination of a DNA mimic of 28S ribosomal RNA [26]. This methodwas also used to detect the enzymatic activity of ricin in swabs from a public health investigation [27],as well as to detect enzymatically active ricin spiked into a variety of beverages with a reported limitof detection of approximately 64 ng/mL [21]. This method was later adapted for analysis of ricin in ashorter timeframe [30]. Although this analysis was not used in the EQuATox study, it remains a viablealternative for evaluation of the enzymatic activity of ricin provided that the specificity limitations ofthe assay are well understood.Toxins 2015, 7 10

Figure 3. Mass spectrum of the reaction of an RNA substrate with ricin; the peak at

m/z 4535.8 corresponds to the mass of the intact substrate and the peak at m/z 4418.7

corresponds to depurination of the RNA substrate by ricin.

4.3. MS/MS Analysis of Ricin Activity Measurements

Upon depurination of a 28S ribosomal mimic by ricin, free adenine is released from the GAGA

tetraloop. Rather than monitoring the mass shift of the RNA mimic, this release of free adenine can be

monitored by MS/MS techniques. This technique was first reported in 2007, using a triple quadrupole

mass spectrometer measuring adenine and its specific fragment ions [31]. This technique resulted in the

ability to detect ricin at levels as low as 0.1 ng/mL following concentration of the toxin from

0.5 mL of sample [31]. Recently, this method was used to examine the enzymatic activity of ricin in

eighteen cultivars from R. communis [22]. The approach was used by at least one laboratory as part of

the EQuATox study, and the ricin and/or RCA120 protein were found in samples S2, S3, S4, S5, S6, S8

and S9 as seen in Table 1. Sample S7 contained the lowest ricin concentration of all the samples, and

due to sample volume limitations, ricin activity was not observed in that sample.

An optional challenge of the EQuATox project was to rank the functional activity of three of the

samples, S1, S3 and S5, using the designations of “highest”, “intermediate” and “lowest” functional

activity. Through analysis of the released adenine, LC-MS/MS in at least one laboratory correctly

identified sample S1 as the least in functional activity followed by sample S5 as intermediate functional

activity and then sample S3 as highest functional activity. All three of these samples consisted of the

same buffer matrix, but contained either no toxin (S1), 445 μg/L of RCA120 (S5), or 504 μg/L of ricin (S3).

Even though the concentrations of toxin in samples S3 (containing ricin) and S5 (containing RCA120)

were similar, their functional activities were correctly assigned. This analysis also used immunoaffinity

enrichment with antibodies to both ricin and RCA120.

5. Addition of Affinity Techniques Assists MS and MS/MS Analysis of Ricin

Although mass spectrometric measurements of ricin can yield useful information, a purification and

enrichment step prior to MS or MS/MS analysis assists in controlling unwanted background

interferences and lowers the limit of detection. The increase in the concentration of ricin in the sample

makes it possible to use MS identification techniques to unambiguously confirm results obtained by

highly sensitive ELISA and activity measurements in extremely dilute samples.

Figure 3. Mass spectrum of the reaction of an RNA substrate with ricin; the peak at m/z 4535.8corresponds to the mass of the intact substrate and the peak at m/z 4418.7 corresponds to depurinationof the RNA substrate by ricin.

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4.3. MS/MS Analysis of Ricin Activity Measurements

Upon depurination of a 28S ribosomal mimic by ricin, free adenine is released from the GAGAtetraloop. Rather than monitoring the mass shift of the RNA mimic, this release of free adenine can bemonitored by MS/MS techniques. This technique was first reported in 2007, using a triple quadrupolemass spectrometer measuring adenine and its specific fragment ions [31]. This technique resulted inthe ability to detect ricin at levels as low as 0.1 ng/mL following concentration of the toxin from0.5 mL of sample [31]. Recently, this method was used to examine the enzymatic activity of ricin ineighteen cultivars from R. communis [22]. The approach was used by at least one laboratory as part ofthe EQuATox study, and the ricin and/or RCA120 protein were found in samples S2, S3, S4, S5, S6, S8and S9 as seen in Table 1. Sample S7 contained the lowest ricin concentration of all the samples, anddue to sample volume limitations, ricin activity was not observed in that sample.

An optional challenge of the EQuATox project was to rank the functional activity of threeof the samples, S1, S3 and S5, using the designations of “highest”, “intermediate” and “lowest”functional activity. Through analysis of the released adenine, LC-MS/MS in at least one laboratorycorrectly identified sample S1 as the least in functional activity followed by sample S5 as intermediatefunctional activity and then sample S3 as highest functional activity. All three of these samplesconsisted of the same buffer matrix, but contained either no toxin (S1), 445 µg/L of RCA120 (S5),or 504 µg/L of ricin (S3). Even though the concentrations of toxin in samples S3 (containing ricin)and S5 (containing RCA120) were similar, their functional activities were correctly assigned. Thisanalysis also used immunoaffinity enrichment with antibodies to both ricin and RCA120.

5. Addition of Affinity Techniques Assists MS and MS/MS Analysis of Ricin

Although mass spectrometric measurements of ricin can yield useful information, a purificationand enrichment step prior to MS or MS/MS analysis assists in controlling unwanted backgroundinterferences and lowers the limit of detection. The increase in the concentration of ricin in the samplemakes it possible to use MS identification techniques to unambiguously confirm results obtained byhighly sensitive ELISA and activity measurements in extremely dilute samples.

5.1. Immunoaffinity as a Purification Technique

Addition of immunoaffinity prior to ricin mass spectrometric analysis was first reported in2007 where the authors used this technique prior to LC-MS/MS analysis of adenine released asa result of ricin’s enzymatic activity [31]. Since then, many studies have reported on the useof immunoaffinity prior to mass spectrometric analysis, including MS of the toxin itself [19,32],MS/MS of the toxin [21,22,26,27], MS of ricin’s activity [21,26,27] and MS/MS of ricin’s activity [22].Several successful EQuATox mass spectrometric analyses used immunoaffinity capture prior to massspectrometry, such as the data presented in Table 1. Both the purification and concentration of thericin by immunoaffinity was critical to these mass spectrometric measurements, especially in complexsample matrices.

5.2. Carbohydrate Affinity as a Purification Technique

Other techniques for purification/concentration prior to mass spectrometric analysis of ricininvolved the ability of ribosome-inactivating proteins such as ricin to bind galactose-terminatedglycolipids and glycoproteins on the cell surface. Galactose affinity has commonly been usedfor large-scale purification of ricin and other RIP-II toxins from plant material [33,34]. Adaptionto analytical scale purification was reported in 2011, with the use of lactose immobilized tomonolithic silica to extract ricin prior to MS/MS analysis [35]. Another report in 2015 used agalactose-terminated ligand bound to chromatographic resin prior to MS/MS analysis of ricin andother ribosome-inactivating proteins [28]. One laboratory successfully used this affinity techniquein all of the EQuATox samples except for the sample containing spiked milk as the lactose content,

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which prevents lectin binding and requires the use of immunoaffinity purification. Thus, for mostsample matrices, galactose affinity remains a viable and less expensive alternative to immunoaffinityand has the advantage of enrichment of multiple ribosome-inactivating proteins.

6. Quantification of Ricin and Its Enzymatic Activity

6.1. Transforming a Qualitative MS Assay into a Quantitative Assay

Mass spectrometric assays for ricin can be transformed from qualitative to quantitative throughthe addition of an internal standard into the samples and into a calibration curve, containing knownamounts of ricin. In most cases, the internal standard is an isotopically-labeled version of the analyte:either an isotopically-labeled tryptic peptide in the case of MS or MS/MS analyses of the ricin toxin oran isotopically-labeled version of adenine in MS/MS assays to detect ricin’s enzymatic activity. Theuse of an isotopically-labeled version of the native analyte ensures that the chemical properties ofthe internal standard which determine chromatographic retention time and ionization potential arethe same as the native analyte, yet the internal standard can easily be differentiated from the nativeanalyte due to the increase in mass.

6.2. Quantification of Ricin by MS/MS

The amount of ricin in the EQuATox samples was reported using MS/MS on the toxin itselfby one laboratory. The qualitative analyses of the EQuATox samples showed that samples S3, S4,S6, S8 and S9 contained ricin. Table 4 lists the concentrations of ricin, using a targeted LC-MS/MSmethod with immunoaffinity purification and peptides underlined in Figure 1 and listed in Table 1,found in samples S3, S4, S6, S7, S8 and S9 along with the nominal concentrations. One measure ofaccuracy is known as a z-score, which indicates how many standard deviations a particular value isfrom the mean. Z-scores in the range of ´2 to +2 are considered to be acceptable. The z-scores are alsolisted in Table 4, and four of the six ricin measurements (Table 4) were within this acceptable range,and only one score lay outside of +/´3, indicating room for improvement. The one measurementin the unacceptable range was for the one solid sample, the fertilizer, and it is likely that an error inthe creation of a liquid extract from the fertilizer caused the quantification error. Additionally, theone measurement outside of the ´2 to +2 range was below the limit of detection. Nonetheless, theresults are quite good taking into account that different reference materials and a technically differentmethod were used to compare to the reference materials and the method used in the organizinglaboratory, respectively.

Table 4. Quantitative results for MS/MS analysis of EQuATox samples for ricin.

Sample Nominal Conc. Unit Obs Conc. #1 Obs Conc. #2 Z-Score

S3 504 µg/L 590 693 0.89S4 473 µg/L 641 641 1.85S6 589508 µg/L 507,500 526,600 ´0.48S7 0.414 µg/L 0.22 0.10 ´2.5S8 484 µg/L 497 659 0.54S9 306 mg/kg 15.3 12.5 ´3.66

6.3. Quantification of RCA120 by MS/MS

Samples S2, S5 and S9 contained RCA120, and one laboratory attempted quantification of theRCA120 by mass spectrometric analysis, using the peptides underlined in Figure 1 and listed inTable 2. The results of the analyses were that sample S2 contained an average of 776,000 µg/L,sample S5 contained 997 µg/L and sample S9 contained an average of 4 mg/kg of RCA120. Theactual concentrations were revealed to be 572,851 µg/L for sample S2, 445 µg/L for sample S5 and42 mg/kg for sample S9. Only the measurement for sample S2 had a z-score within +/´2. This is

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likely due in part to the emphasis of assays on ricin rather than RCA120, as fewer ions were used tomonitor RCA120 as compared to ricin, and the general optimization of the assay for the quantificationof ricin. Another source of error could be the difficulty in obtaining reference materials of purifiedricin, which do not contain RCA120 and vice versa, as there are no known cultivars, which produceeither toxin exclusively. The purity of reference materials used in the EQuATox study was >97% forricin and >99% for RCA120 [36]. Other commercial sources of ricin and RCA120 such as the materialused for the calibration curves contain higher levels of contamination from RCA120 and ricin [20].Therefore, methods, which are sensitive, could yield a positive result for RCA120, for instance,particularly in the presence of a high concentration of ricin. Highly accurate and reproduciblequantification requires that a reliable and well-characterized reference standard be employed.

7. Summary

Through the EQuATox study, it has been demonstrated that mass spectrometric measurementsof ricin play a vital role in correct identification of ricin in complex matrices. Some mass spectrometricmeasurements of ricin serve as accessory methods which, when used in combination with othermethods, can yield information helpful in determining the presence of ricin either through amolecular weight measurement designed to differentiate ricin from RCA120 or through an enzymaticactivity measurement of a ribosome-inactivating protein as seen in Figure 3. Other mass spectrometricmeasurements, such as MS/MS measurements, can stand alone as a definitive identification of ricin,proving the existence of the unique amino acid sequence of ricin.

In analyses with limited sample volume and the desire to perform multiple mass spectrometricmeasurements, a valid approach would be to divide each sample into separate aliquots forenzymatic activity measurements and structural measurements, perhaps putting more emphasison the enzymatic activity measurements for public health purposes or highlighting the structuralmeasurements for forensic purposes. For structural measurements to be used as definitiveidentification of ricin, MS/MS detection of two peptides specific and unique to ricin, such as some ofthe peptides listed in Table 1, can be used for positive detection.

EQuATox is the first international program to investigate qualitative and quantitative results foran assortment of ricin detection methods using well-characterized analytical standards. The resultsfrom EQuATox will become an important basis for international discussions on the exact needs forricin quantification for security and public health and what reference standards are necessary in orderto meet patient, public health and law enforcement needs. Mass spectrometry can play a critical rolein the correct identification of ricin in complex matrices. With its ability to yield the functional activitymeasurements critical for public health needs and its ability to accurately differentiate between ricinand RCA120 needed for forensics reasons, mass spectrometry is a useful tool for ricin detectionencompassing a wide variety of detection goals.

8. Experimental Section

8.1. Targeted MS/MS Qualitative and Quantitative Analysis

Targeted MS/MS qualitative analysis proceeded as described previously [22]. Essentially,magnetic beads coated with antibodies to ricin and RCA120 were added to the sample and allowedto bind toxin if present. Following bead washing, the beads were digested with trypsin andthe subsequent peptides were analyzed by LC-MS/MS on a triple quadrupole mass spectrometer,monitoring for peptides underlined in Figure 1 and listed in Table 1. Transitions monitoredwere previously described [22]. Quantitative analyses were performed in a similar fashion usingisotopically-labeled tryptic peptides and transitions as described earlier [22].

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8.2. Accurate Mass LC-MS and MS/MS Qualitative Analysis

Non-targeted LC-MS and MS/MS analyses were performed as described previously [28]. Briefly,sample aliquots (10–200 µL depending on quantification results obtained by ELISA) were purifiedand concentrated by galactose affinity chromatography. Galactose-binding proteins were digestedwith trypsin without prior reduction and alkylation. Sample S4 was suspected to contain milk,in which lactose interferes with the galactose affinity purification. Therefore, aliquots of S4 wereimmunopurified using a biotinylated polyclonal antibody to ricin and RCA120 coupled to DynabeadsStreptavidin T-1 magnetic beads.

The digests were analyzed by high-resolution accurate mass nanoLC-MS on a QTOF instrument(Waters Corporation, Milford, MA, USA), and the retention times and masses of the peptides werecompared with those pf ricin and RCA120 reference digest. The identity of diagnostic peptides wasconfirmed by nanoLC-MS/MS analysis using time-scheduled precursor ion selection and comparisonwith the corresponding peptides in the reference digests.

8.3. MS/MS of Enzymatic Activity of Ricin

The enzymatic activity of ricin was examined as described previously [22,31]. Basically,following immunocapture of ricin or RCA120 from the sample and bead washing, the toxin onthe beads was allowed to interact with RNA substrate CGCGCGAGAGCGCG or DNA substrateGCGCGAGAGCGC, resulting in the release of free adenine in the presence of ricin or RCA120.The presence of free adenine was monitored using triple quadrupole mass spectrometry using thetransitions of m/z 136 to 119, 136 to 92 and 136 to 65 as described previously [22,31]. Quantificationof the enzymatic activity of ricin and RCA120 was also accomplished as previously described [22,31]using either 15N2-1,3-adenine or 13C-labeled adenine as an internal standard.

Acknowledgments: The findings and conclusions in this report are those of the authors and do not necessarilyrepresent the official position of the Centers for Disease Control and Prevention.

Author Contributions: All authors analyzed data and were involved in the preparation and writing of the paper.

Conflicts of Interest: The authors declare no conflict of interest.

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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an openaccess article distributed under the terms and conditions of the Creative Commons byAttribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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